Human telomeric protein TRF2 associates with genomic double-strand breaks as an early response to DNA damage.

DNA damage surveillance networks in human cells can activate DNA repair, cell cycle checkpoints and apoptosis in response to fewer than four double-strand breaks (DSBs) per genome. These same networks tolerate telomeres, in part because the protein TRF2 prevents recognition of telomeric ends as DSBs by facilitating their organization into T loops. We now show that TRF2 associates with photo-induced DSBs in nontelomeric DNA in human fibroblasts within 2 s of irradiation. Unlike gammaH2AX, a common marker for DSB damage, TRF2 forms transient foci that colocalize closely with DSBs. The TRF2 DSB response requires the TRF2 basic domain but not its Myb domain and occurs in the absence of functional ATM and DNA-PK protein kinases, MRE11/Rad50/NBS1 complex and Ku70, WRN and BLM repair proteins. Furthermore, overexpression of TRF2 inhibits DSB-induced phosphorylation of ATM signaling targets. Our results implicate TRF2 in an initial stage of DSB recognition and processing that occurs before association of ATM with DSBs and activation of the ATM-dependent DSB response network.

A common molecular mechanism underlies two phenotypically distinct 17p13.1 microdeletion syndromes.

DNA copy-number variations (CNVs) underlie many neuropsychiatric conditions, but they have been less studied in cancer. We report the association of a 17p13.1 CNV, childhood-onset developmental delay (DD), and cancer. Through a screen of over 4000 patients with diverse diagnoses, we identified eight probands harboring microdeletions at TP53 (17p13.1). We used a purpose-built high-resolution array with 93.75% breakpoint accuracy to fine map these microdeletions. Four patients were found to have a common phenotype including DD, hypotonia, and hand and foot abnormalities, constituting a unique syndrome. Notably, these patients were not affected with cancer. Moreover, none of the TP53-deletion patients affected with cancer (n = 4) had neurocognitive impairments. DD patients have larger deletions, which encompass but do not disrupt TP53, whereas cancer-affected patients harbor CNVs with at least one breakpoint within TP53. Most 17p13.1 deletions arise by Alu-mediated nonallelic homologous recombination. Furthermore, we identify a critical genomic region associated with DD and containing six underexpressed genes. We conclude that, although they overlap, 17p13.1 CNVs are associated with distinct phenotypes depending on the position of the breakpoint with respect to TP53. Further, detailed characterization of breakpoints revealed a common formation signature. Future studies should consider whether other loci in the genome also give rise to phenotypically distinct disorders by means of a common mechanism, resulting in a similar formation signature.

Defining and Reporting on Critical Values in Genetics: A Laboratory Survey.

BACKGROUND: Although an obvious critical value in metabolic genetics would be ammonia, it is more challenging to define critical values in molecular genetics and cytogenetics. The objective of this study was to survey genetic laboratories in Ontario, Canada, to determine whether different centers considered similar results as critical and thus potentially deserving of a different reporting process. METHODS: An online 11-question survey was emailed to Ontario laboratory directors, and the results were analyzed. RESULTS: The response rate was 82% (9/11). Cytogenetics and molecular genetics services were each provided by 7 of the 9 centers, with 3 centers providing biochemical/metabolic genetics services and 1 providing maternal marker serum screening services. The case type (e.g., prenatal, newborn, or expedited by the ordering physician) was one factor. Quantitative fluorescence PCR for autosomal aneuploidy, pathogenic variants in both prenatal and postnatal settings, and oncological results were considered critical cytogenetics results. Pathogenic prenatal cases, indeterminate results, and unexpected results were considered more critical for molecular genetics. Critical results were more likely to prompt a telephone call or email to the ordering physician. CONCLUSION: Ontario genetics laboratories tended to have similar reporting processes for critical results. Both the types of cases and the pathogenicity of the result define what values are considered critical.

Recurrent focal copy-number changes and loss of heterozygosity implicate two noncoding RNAs and one tumor suppressor gene at chromosome 3q13.31 in osteosarcoma.

Osteosarcomas are copy number alteration (CNA)-rich malignant bone tumors. Using microarrays, fluorescence in situ hybridization, and quantitative PCR, we characterize a focal region of chr3q13.31 (osteo3q13.31) harboring CNAs in 80% of osteosarcomas. As such, osteo3q13.31 is the most altered region in osteosarcoma and contests the view that CNAs in osteosarcoma are nonrecurrent. Most (67%) osteo3q13.31 CNAs are deletions, with 75% of these monoallelic and frequently accompanied by loss of heterozygosity (LOH) in flanking DNA. Notably, these CNAs often involve the noncoding RNAs LOC285194 and BC040587 and, in some cases, a tumor suppressor gene that encodes the limbic system-associated membrane protein (LSAMP). Ubiquitous changes occur in these genes in osteosarcoma, usually involving loss of expression. Underscoring their functional significance, expression of these genes is correlated with the presence of osteo3q13.31 CNAs. Focal osteo3q13.31 CNAs and LOH are also common in cell lines from other cancers, identifying osteo3q13.31 as a generalized candidate region for tumor suppressor genes. Osteo3q13.31 genes may function as a unit, given significant correlation in their expression despite the great genetic distances between them. In support of this notion, depleting either LSAMP or LOC285194 promoted proliferation of normal osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1. Moreover, genetic deletions of LOC285194 or BC040587 were also associated with poor survival of osteosarcoma patients. Our findings identify osteo3q13.31 as a novel region of cooperatively acting tumor suppressor genes.

The Bloom syndrome helicase BLM interacts with TRF2 in ALT cells and promotes telomeric DNA synthesis.

Telomerase-negative immortalized human cells maintain telomeres by alternative lengthening of telomeres (ALT) pathway(s), which may involve homologous recombination. We find that endogenous BLM protein co-localizes with telomeric foci in ALT human cells but not telomerase positive immortal cell lines or primary cells. BLM interacts in vivo with the telomeric protein TRF2 in ALT cells, as detected by FRET and co-immunoprecipitation. Transient over-expression of green fluorescent protein (GFP)-BLM results in marked, ALT cell-specific increases in telomeric DNA. The association of BLM with telomeres and its effect on telomere DNA synthesis require a functional helicase domain. Our results identify BLM as the first protein found to affect telomeric DNA synthesis exclusively in human ALT cells and suggest that BLM facilitates recombination-driven amplification of telomeres in ALT cells.